My broad and long-term objective is to develop murine indicator lines that allow us to selectively determine alterations of signal transduction in vascular endothelium in an inexpensive, high-throughput format. Vascular endothelium plays a number of critical roles in normal development and homeostasis of the cardiovascular system and in the pathogenesis of many human cardiovascular diseases. Vascular endothelium senses a variety of chemical and physical changes occurring in its microenvironment. Aberrant regulation of this sensing mechanism, called signal transduction, in vascular endothelium often leads to dysfunction of this cell type, and is manifested as many forms of diseases. The mouse is an excellent animal model to study human diseases. In past years, numerous mutant mice have been generated by either targeted or random mutagenesis, and more mutants will be generated at an accelerated rate. Therefore, development of inexpensive high-throughput phenotypic screening methods will be essential for us to catch up with the rate at which mutant mice are generated. In this proposal, I focus on the development of a method that allows us to determine biochemical alterations of specific signal transduction pathways selectively in vascular endothelial cells. This goal will be accomplished with the following six specific aims: (1) To develop mouse indicator lines that allow the measurement of alterations of specific signal transduction pathways selectively in vascular endothelial cells. (2) to establish temporal and spatial maps of activation and inactivation of each of the signal transduction pathway in vascular endothelium by using the indicator lines. (3) To establish a biochemical assay in a high-throughput format by using tissues from the indicator lines. (4) To generate these indicators in various genetic backgrounds to allow immediate crossing to the existing and future mutant mice. (5) To generate embryonic stem cell lines from the indicator lines. (6) To establish a web site to streamline the dissemination oft he reagents, protocols, and information established through this project. Availability of our high-throughput screening method should lead to our further understanding development and physiological function of the cardiovascular system. Furthermore, the knowledge gained through the screening may result in the emergence of new concepts related to the regulation of cardiovascular development and function. (End of Abstract.)